1. Technical Field
This present invention relates to an image processing method of two-photon structured illumination point scanning microscopy, more particularly, to an image processing method of two-photon structured illumination point scanning microscopy that can be used in the observation of biological cell sample.
2. Description of Related Art
In our living environment, it is usually filled with all kinds of bacteria, viruses, or other factors that might endanger human health or environment. Therefore, biomedical research is always an important target in the scientific development process. In the research process, microscopic image technology plays an important role. Among them, microscopic image technology based on laser as a light source has further enhanced its resolution; therefore, it has provided a powerful tool for biomedical research field.
However, the prior art technology using laser as the light source for microscopic image is the so-called “single-photon microscopy”, which adopts laser light with each photon energy sufficient to let the molecule in the sample-under-test make a transition to excited state.
As shown in FIG. 1, since the energy of each photon in the optical beam 11 of a laser light source 10 used in single-photon microscopy is sufficient to let a molecule in a sample-under-test 20 get excited, then make a transition back to a ground state from an excited state and release photon hv and get observed or recorded. Therefore, within the illumination path of the optical beam 11 emitted by the laser light source 10, all the molecules will have chance to get excited, then make a transition back to the ground state and release photon hv and get observed and recorded. A great part of the observed or recorded image signal is not generated by molecules on the focal plane P0 that the observer is originally planning to observe. Thus, the resolution of the microscopic image in the axial direction is reduced.
In order to solve the bad axial resolution issue of single-photon microscopy, “Two-Photon Scanning Microscopy (TPSM)” is thus proposed. The largest difference between two-photon microscopy and single-photon microscopy is: In two-photon scanning microscopy, the energy of each photon in the laser light source used is only half of the energy needed for the sample's molecule to make a transition from the ground state to the excited state. Under such condition, to excite the molecule in the sample to make a transition to the excited state, it is necessary to let the same molecule, at the same time, receive two incident photons and absorb them. That is, it is necessary to have two photons, at the same time, incident on the same spatial location. Therefore, only the molecules of the sample located at the focal spot of the laser light source will have higher probability to satisfy simultaneously temporal and spatial location limit and get excited. Consequently, the resolution of the obtained microscopic image in the axial direction will be greatly enhanced. However, due to the physical characteristic limit of optical component used to focus laser light source, especially the diffraction limit of the optical component, it will lead to the difficulty of the laser light source to be focused on smaller area. Therefore, even if two-photon scanning microscopy is adopted, the improvement on the resolution of the microscopic image is still limited.
Hence, how to improve or solve effectively the above problems so as to obtain microscopic image of higher resolution has become important target for related researchers or suppliers.